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Abstract

Glomerular hypertrophy and hyperfiltration are the two major pathological characteristics of the early stages of diabetic nephropathy (DN), which are respectively related to mesangial cell (MC) proliferation and a decrease in calcium influx conducted by canonical transient receptor potential cation channel 6 (TRPC6). The marked increase in the production of reactive oxygen species (ROS) induced by hyperglycemia is the main sponsor of multiple pathological pathways in DN. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is an important source of ROS production in MCs. Astragaloside IV (AS‑IV) is an active ingredient of Radix Astragali which has a potent antioxidative effect. In this study, we aimed to investigate whether high glucose (HG)‑induced NADPH oxidase activation and ROS production contribute to MC proliferation and the downregulation of TRPC6 expression; we also wished to determine the effects of AS‑IV on MCs under HG conditions. Using a human glomerular mesangial cell line, we found that treatment with AS‑IV for 48 h markedly attenuated HG‑induced proliferation and the hypertrophy of MCs in a dose‑dependent manner. The intracellular ROS level was also markedly reduced following treatment with AS‑IV. In addition, the enhanced activity of NADPH oxidase and the expression level of NADPH oxidase 4 (Nox4) protein were decreased. Treatment with AS‑IV also inhibited the phosphorylation level of Akt and IκBα in the MCs. In addition, TRPC6 protein expression and the intracellular free calcium concentration were also markedly reduced following treatment with AS‑IV under HG conditions. These results suggest that AS‑IV inhibits HG‑induced mesangial cell proliferation and glomerular contractile dysfunction through the NADPH oxidase/ROS/Akt/nuclear factor‑κB (NF‑κB) pathway, providing a new perspective for the clinical treatment of DN.

Introduction

Diabetic nephropathy (DN) is one of the main chronic
complications of diabetes mellitus (DM) associated with
capillaries, often leading to chronic renal failure and end-stage
renal disease (1). Although the
precise pathogenesis of DN has not been elucidated, a number of
studies have demonstrated that glomerular hyperfiltration and
mesangial expansion are the two characteristic pathophysiological
changes occurring during the early stages of DN (2,3).

Mesangial cells (MCs), located within glomerular
capillary loops, play an important role in the regulation of
glomerular hemodynamics due to their contractile function (4). There is increasing evidence
indicating that hyperglycemia directly stimulates MCs, which
subsequently results in mesangial contractile dysfunction and
glomerular hyperfiltration (5,6).
The hypocontractility of MCs is closely related to the reduced
calcium influx (7,8).

Canonical transient receptor potential cation
channels (TRPCs), as members of the transient receptor potential
(TRP) superfamily, are Ca2+ permeable cation channels
widely expressed in a series of tissues and cells (9,10).
The TRPC family includes seven related members, designated as
TRPC1–7 (11). Among these, TRPC6
is closely associated with kidney disease (12,13). Möller et al (14) found that TRPC6 exists throughout
the glomerulus in kidney tissues, particularly in MCs.
Additionally, it has been dcemonstrated that hyperglycemia
downregulates the expression of TRPC6 protein, which results in a
decrease in intracellular calcium, leading to impaired MC
contractile response and glomerular hyperfiltration (15). However, the underlying molecular
mechanisms of TRPC6 protein downregulation in MCs have not yet been
fully elucidated.

Hyperglycemia can also cause the abnormal
proliferation of MCs in DN, leading to glomerular hypertrophy and
fibrosis (16). Clinical and
animal experiments have demonstrated an increase in the production
of reactive oxygen species (ROS) in MCs, as the direct result of
chronic exposure to high glucose (HG) (17,18). The overproduction of ROS can
transduce and amplify glucose signaling, playing a key role in MC
proliferation (19). ROS can also
modulate Ca2+ channels by activating various signaling
cascades (20).

In the kidneys, particularly in MCs, nicotinamide
adenine dinucleotide phosphate (NADPH) oxidase is the predominant
source of ROS (21). The
phagocyte NADPH oxidase consists of two membrane-associated
subunits, p22phox and gp91phox, and at least
four cytoplasmic components, p47phox,
p67phox, p40phox and the small GTPase, Rac.
NADPH oxidase in the resting state becomes activated upon
interaction between the catalytic core (membrane-associated
subunits) and the cytosolic regulatory subunits (22). There are six homologues of
phagocytic gp91phox proteins expressed by distinct
non-phagocytic cells (23). It
has been reported that NADPH oxidase 4 (Nox4) is the key subunit of
NADPH oxidase expressed in MCs, and Nox4-derived ROS is the major
contributor to renal morphological changes and functional
abnormalities in DN (24).

Akt, also known as protein kinase B (PKB), belonging
to serine/threonine kinase family members, is one of the downstream
effectors of phosphoinositide 3-kinase (PI3K) which participates in
numerous signaling pathways involved in diverse physiological
processes, including glucose metabolism, protein synthesis, cell
proliferation, cell apoptosis (25,26). Kim et al reported that Akt
was activated in renal damage in streptozotocin-induced diabetic
mice (27). It has also been
demonstrated that Akt is an important mediator of MC proliferation,
and can be regulated by ROS (28).

Akt is not only the downstream signaling molecule of
PI3K, but also the major upstream element in the activation of
nuclear factor-κB (NF-κB). Akt promotes the transcriptional
activity of NF-κB through a variety of mechanisms and the signaling
cascade eventually leads to cell proliferation and migration
(29). A number of studies have
demonstrated that NF-κB is activated in MCs by hyperglycemia, and
plays a crucial role in the progression of DN (30,31). In addition, a recent study found
that ROS is the important messenger in the NF-κB signaling pathway
(32). Of note, the promoter
region of TRPC6 has NF-κB binding sites (33). Therefore, we hypothesized that the
redox-sensitive NF-κB participates in the downregulation of TRPC6
in DN. Thus, we aimed to explore whether NADPH oxidase-derived ROS
is involved in HG-induced cell proliferation and the downregulation
of TRPC6 in MCs through the Akt/NF-κB pathway.

Astragaloside IV (AS-IV,
3-O-β-D-xylopyranosyl-6-O-β-D-glucopyranosylcycloastragenol), a
purified small molecular saponin, is one of the main active
ingredients of Radix Astragali, which has been reported to possess
comprehensive biological properties, including antioxidant,
anti-inflammatory, immunoregulatory and anti-aging properties and
to improve intellectual development (34). A recent studies indicated that
AS-IV ameliorates proteinuria in rats with adriamycin nephropathy
(35). It has also been reported
that AS-IV significantly inhibits renal oxidative stress and
apoptosis in STZ-induced diabetic rats (36). However, the protective effects and
the precise mechanisms of action of AS-IV on oxidative
stress-induced injury in MCs under HG conditions have not yet been
fully elucidated.

The present study aimed to investigate the effects
of AS-IV on HG-induced MC proliferation and the downregulation of
TRPC6 through a mechanism associated with the inhibition of NADPH
oxidase-mediated ROS production, Akt and NF-κB activation, in an
attempt to provide a novel therapeutic approach for the treatment
of DN.

AS-IV was purchased from Nanjing Zelang Medical
Technology Co., Ltd. (Nanjing, China; purity >98%, HPLC). The
chemical structure of AS-IV
(C41H68O14; molecular weight, 784)
is depicted in Fig. 1. AS-IV was
dissolved in DMSO to the concentration of 25 μmol/ml as a stock
solution. The stock solution was diluted with DMEM into AS-IV
solutions according to the respective group when used and the final
DMSO concentration did not exceed 0.5% (v/v).

MC culture

The human mesangial cell line (HMC) was obtained
from the Modern Analysis and Testing Center of Central South
University (Changsha, China), and maintained in normal DMEM (5.6 mM
glucose) supplemented with 10% FBS (v/v), 100 μ/ml penicillin and
100 μg/ml streptomycin at 37°C in an atmosphere containing 5%
CO2. HG treatment was performed by culturing the cells
in DMEM containing 25 mM glucose for 48 h.

Cell proliferation assay

Cells were seeded at a density of 1×104
cells/well in 96-well plates. When the cell confluence reached at
70–80%, the growth medium was replaced with DMEM containing 5.6 mM
glucose and 0.5% FBS. After 24 h, the quiescent cells were treated
with the indicated concentrations of glucose together with various
concentrations of AS-IV (5, 10, 25, 50 and 100 μM) or 0.5% DMSO
(vehicle control) for 48 h. The osmotic control medium was made by
supplementing normal DMEM with 24.5 mM MA. Following incubation
with the above-mentioned compounds, cell proliferation was
determined by MTT assay (37).
The absorbance was measured at 490 nm using a SpectraMax 190
Microplate Reader (Molecular Devices, Sunnyvale, CA, USA).

Measurement of total protein to cell
count ratio

The ratio of the total protein content to the cell
number is a well-established measure of cellular hypertrophy
(38). The MCs were seeded into
6-well plates and were synchronized into quiescence in DMEM
containing a normal glucose concentration and 0.5% FBS for 24 h.
The cells were then stimulated with HG and treated with various
concentrations of AS-IV (5, 10, 25, 50 and 100 μM) or 0.5% DMSO
(vehicle control) for 48 h. Following incubation, the cells were
washed twice with PBS and trypsinized. A small aliquot of the cells
was used for cell counting by a hemocytometer. The remaining cells
were lysed in RIPA buffer (Beyotime, Haimen, China), and the total
protein content was measured by a protein quantitative reagent
kit-BCA method (Beyotime). The total protein content to the cell
count ratio was expressed as microgram protein per 104
cells.

Detection of intracellular ROS
generation

The generation of ROS was detected using the
membrane permeable indicator, DCFH-DA. The cells were seeded in
24-well plates at a density of 1×105 cells/well. After
being synchronized, the cells were cultured in DMEM containing 5.6
or 25 mM glucose with or without various concentrations of AS-IV
(25, 50 and 100 μM) or tempol (100 μM) for 48 h, were then loaded
with 10 μM DCFH-DA in serum-free DMEM containing 5.6 or 25 mM
glucose at 37°C for 30 min in the dark, and the cell culture plate
was shaken every 5 min and washed three times with PBS in order to
remove residual probes. Subsequently, intracellular ROS production
were observed under a fluorescence microscope (Nikon, Tokyo, Japan;
excitation at 488 nm, emission at 525 nm). The mean fluorescence
intensity for each group of cells was determined using the
Image-Pro Plus 6.0 analysis system (MediaCybernetics, Rockville,
MD, USA).

NADPH oxidase activity assay

NADPH oxidase activity in the MCs was measured using
the cell NADPH oxidase colorimetric assay kit (GenMed Scientifics
Inc.) according to the manufacturer’s instructions. Briefly, the
cells grown in DMEM containing 5.6 or 25 mM glucose in the presence
or absence of AS-IV (25, 50 and 100 μM) or DPI (10 μM) for 48 h
were washed twice in PBS and scraped from the plate followed by
centrifugation at 12,000 g, 4°C, for 3 min, and suspended in PBS,
followed by incubation with 250 μM NADPH. NADPH consumption was
monitored by a decrease in absorbance at 340 nm for 5 min using a
SpectraMax 190 Microplate Reader (Molecular Devices). NADPH oxidase
activity was defined as picomoles per liter of substrate per minute
per milligram of protein.

Western blot analysis

The cells were lysed with cold lysis buffer
containing protease inhibitors. Equal amounts of protein extracts
were fractionated by SDS-PAGE and then transferred onto
polyvinylidene difluoride (PVDF) membranes (Millipore Corp.,
Bedford, MA, USA). After being blocked with 5% non-fat milk in
Tris-buffered saline with Tween-20 (TBST, pH 7.6) for 1 h at room
temperature, the membranes were incubated with the indicated
primary antibodies (Nox4, 1:200; Akt, 1:500; phospho-Akt, 1:500;
IκBα, 1:1,000; phospho-IκBα, 1:200; TRPC6, 1:1,000 and β-actin,
1:1,000) overnight at 4°C. The membranes membranes were rinsed
three times with TBST and incubated with the respective secondary
antibodies (1:10,000 dilutions of each antibody) for 1 h at room
temperature. The protein bands were visualized with SuperSignal
West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific
Inc., Rockford, IL, USA) and captured using a Bioshine ChemiQ 4600
Mini Chemiluminescence imaging system (Ouxiang, Shanghai, China).
The optical density of each band was quantified using ImageJ
software (NIH, Bethesda, MD, USA) and normalized to the intensity
of β-actin.

Fluorescence measurement of
[Ca2+]i

Measurements of the intracellular free calcium
concentration were performed using Fluo-3/AM
fluorospectrophotometry. The MCs, grown on 24-well plates, were
loaded with Fluo-3 by incubation for 40 min at 37°C in the dark in
HEPES buffer solution containing 5 μM Fluo-3/AM followed by washing
three times with the same buffer. The cells were then incubated
with Fluo-3-free HEPES buffer for an additional 20 min. Afer being
trypsinized and collected, the fluorescence intensity (F) was
monitored using an F-4600 fluorescence spectrophotometer (Hitachi,
Tokyo, Japan; excitation at 488 nm, emission at 530 nm). The
maximal density (Fmax) was measured using 0.1% Triton
X-100, and the minimum (Fmin) was measured using 20 mM
EGTA. The concentration of calcium was calculated using the
following formula: [Ca2+]i = Kd (F
- Fmin)/(Fmax - F), where Kd
represents the dissociation constant of Fluo-3 and calcium, and its
value is 450 nM.

Statistical analysis

Data are presented as the means ± standard deviation
(SD). Each experiment was repeated at least three times
independently. Statistical analysis was performed using SPSS 13.0
for Windows (SPSS, Inc., Chicago, IL, USA). Statistical differences
between two groups were analyzed by the unpaired Student’s t-test
and differences among multiple groups were analyzed by one-way
ANOVA. In all cases, values of P<0.05 were considered to
indicate statistically significant differences.

Results

Effects of AS-IV on HG-induced MC
proliferation and hypertrophy

To determine the effects of AS-IV on MC
proliferation, the MTT assay and cell counting were employed. As
shown in Fig. 2A and B, HG
significantly stimulated the growth of the MCs in comparison to
normal glucose (NG) conditions (P<0.01). The administration of
AS-IV at the concentration range of 5–100 μM led to a significant
inhibition of cell growth induced by HG. The vehicle control
treated with DMEM containing HG and 0.5% DMSO also showed a
significant increase in the proliferation of MCs. In addition,
AS-IV at the examined concentrations had no effect on the viability
of MCs under NG conditions, which suggested that the inhibitory
effect of AS-IV upon cultured MCs was not due to its cytotoxicity
(Fig. 2C). HG also markedly
stimulated cell hypertrophy, defined as the protein content of MCs
per unit cell number, which was abolished by the AS-IV
administration in a dose dependent manner (Fig. 2D). Unlike HG, the addition of 24.5
mM MA to the medium did not exert an obvious effect on the
proliferation and hypertrophy of MCs as compared with control,
suggesting that the HG-triggered MC proliferation and hypertrophy
were not the results of high osmolality within the medium.

Effect of AS-IV on HG-stimulated ROS
generation in MCs

We examined the effects of AS-IV on HG-induced
intracellular ROS generation by DCFH-DA fluorescent probe assay
using fluorescence microscopy. As demonstrated in Fig. 3, the MCs cultured under HG
conditions for 48 h showed a significant increase in ROS generation
compared to the cells cultured under NG conditions (P<0.01). To
assess the antioxidative effects of AS-IV, we used tempol, a
classic antioxidant, as a positive control. The effects of
HG-induced ROS generation in the MCs were notably decreased by
treatment with AS-IV (25, 50 and 100 μM) or tempol (100 μM), and
AS-IV exerted its antioxidant effects in a dose-dependent
manner.

Effect of AS-IV on HG-mediated NADPH
oxidase activation in MCs

Since NADPH oxidase is the most important source of
intracellular ROS, we used the NADPH oxidase inhibitor, DPI, as a
positive control to assess the effects of AS-IV on HG-induced NADPH
oxidase activity. As shown in Fig.
4A, HG resulted in a significant increase in NADPH activity
that was markedly attenuated by AS-IV (25, 50 and 100 μM) or DPI
(10 μM). As Nox4 is the key membrane subunit of NADPH oxidase
expressed in MCs, we further examined whether AS-IV blocks the
expression level of Nox4 protein. The protein level of the Nox4
subunit was notably upregulated under HG conditions compared to the
control (P<0.01). Treatment with AS-IV (25, 50 and 100 μM) or
DPI (10 μM) for 48 h markedly downregulated the protein expression
level of Nox4 compared to the HG-treated group (Fig. 4B and C).

Inhibitory effects of AS-IV on HG-induced
phosphorylation of Akt and IκBα in MCs

Given the role that Akt and NF-κB signaling plays in
MC growth and proliferation, we examined the effects of AS-IV on
Akt and NF-κB activation. The phosphorylation level of Akt and IκBα
and the protein expression of total Akt and total IκBα were
detected by western blot analysis. It was observed that HG induced
Akt and NF-κB activation, as manifested by the fact that the
relative amount of phosphorylated Akt and IκBα and the degradation
of IκBα were significantly higher compared to the control cells
(Fig. 5). However, treatment with
AS-IV or the PI3K inhibitor, LY294002 (10 μM), effectively
abrogated the HG-induced Akt phosphorylation in the MCs, and the
phosphorylation and degradation level of IκBα was also markedly
inhibited by AS-IV or the IκBα inhibitor, Sul (0.5 mM). AS-IV did
not affect the protein expression level of total Akt in the
HG-cultured cells. These results suggest that AS-IV inhibits
HG-induced Akt and NF-κB activation in MCs.

We also examined whether NADPH oxidase activation in
MCs is dependent on Akt or NF-κB activation. The expression level
of the Nox4 subunit was evaluated in the HG-exposed cells treated
with or without LY294002, DPI, tempol or Sul. LY294002 and DPI
effectively inhibited the HG-induced increase in the expression of
Nox4. However, no significant change in the expression level of
Nox4 was observed in the MCs treated with tempol and Sul (Fig. 6E and F). Taken together, the above
results suggest that NADPH oxidase activation and the PI3K/Akt
pathway may function in parallel or may interplay with each other,
which are upstream of NF-κB in HG-stimulated MCs.

Inhibitory effects of AS-IV on the
HG-induced downregulation of TRPC6 and the reduction in calcium
influx in MCs

TRPC6 is known as a Ca2+-conductive
cation channel and regulates the contractile function of MCs; it
plays a pivotal role during the early stages of HG-induced damage
to MCs (14,15). Thus, in this study, we examined
the effects of AS-IV on the expression level of TRPC6 protein and
the concentration of intracellular free calcium in MCs cultured
under HG conditions. As illustrated in Fig. 7A and C, incubation of the MCs with
HG for 48 h markedly decreased the expression level of TRPC6
protein compared with the cells cultured under NG conditions. The
downregulation of TRPC6 induced by HG was markedly abrogated by
treatment with AS-IV at a concentration of 25 to 100 μM and the
TRPC6 agonist, a diacylglycerol analog,
1-oleoyl-2-acetyl-sn-glycerol (OAG, 100 μM). Furthermore, in the
presence of inhibitors of signaling molecules, such as LY294002,
DPI, tempol and Sul, the HG-induced TRPC6 downregulation was
markedly abolished (Fig. 7B and
D). Ultimately, we detected the intracellular free calcium
concentration in MCs using Fluo-3/AM fluorospectrophotometry. As
shown in Fig. 7E, the HG-induced
reduction in calcium influx in the MCs was also greatly ameliorated
by AS-IV (25, 50 and 100 μM) or OAG (100 μM). These results suggest
that AS-IV protects MCs against contractile dysfunction under HG
conditions by upregulating the TRPC6 protein expression and
increasing Ca2+ influx through the NADPH
oxidase/ROS/Akt/NF-κB signaling pathway.

Discussion

Radix Astragali, the dried root of Astragalus
membranaceus (Fisch.) Bunge, has long been used in
traditional Chinese medicine for the treatment of cardiovascular
diseases and diabetes (39).
Recently, investigations into its active ingredients have attracted
much attention due to the unique pharmacological properties of many
of its constituents (34). AS-IV
is a novel saponin extracted from Radix Astragali, and it has been
reported to ameliorate podocyte apoptosis by attenuating ROS
production and to prevent acute kidney injury by inhibiting
oxidative stress (40,41). In a previous study of ours, we
suggested that AS-IV significantly reduced
H2O2-induced ROS overproduction in MCs
(42). In order to further
demonstrate that treatment with AS-IV can suppress oxidative
stress-induced injury in DN, the present study was designed to
examine the protective effects of AS-IV on the morphological and
functional abnormalities of MCs cultured under hyperglycemic
conditions.

Hyperglycemia, a common condition occurring in
diabetes, markedly increases the production of ROS in MCs (19). The redox imbalance between the
production of ROS and the compensatory response from the endogenous
antioxidant network results in oxidative stress. The interaction of
the excessive ROS generation with biomolecules, such as lipids,
proteins and DNA, can activate a series of cell signaling pathways,
leading to severe kidney injury and dysfunction (43). The most prominent effect is MC
proliferation, which often leads to glomerulosclerosis (GS), renal
fibrosis or even end-stage renal failure (44). NADPH oxidase, a multicomponent
enzyme, is the major source of ROS production in renal cells
(23). NADPH oxidase was
originally found in neutrophils (22). In many non-phagocytic cells, the
Nox family is a homologue of gp91phox, which is the
catalytic subunit of NADPH oxidase, including several types, such
as Nox1, Nox2, Nox3, Nox4 and Nox5 (45). Of these, the Nox4 isoform is
mainly found in MCs (46). The
activity of NADPH oxidase and the expression level of Nox4 protein
are both markedly increased in MCs under HG conditions. In this
study, we found that treatment with AS-IV markedly supressed
HG-induced intracellular ROS generation, as well as MC
proliferation and hypertrophy. Consistently, our experiments
demonstrated that AS-IV markedly attenuated the HG-stimulated NADPH
oxidase activation and the overexpression of Nox4 in MCs. Our
results indicated that there was no obvious direct cytotoxic effect
of AS-IV on MCs. These results provide evidence that AS-IV may
exert an inhibitory effect on HG-induced MC proliferation and
hypertrophy by downregulating Nox4-derived ROS generation.

Since the activation of various cellular molecules,
such as transcription factors, cytokines, hormones and protein
kinases has been reported to contribute to the signal transduction
cascades of DN (47,48), we investigated the mechanisms
through which AS-IV prevents damage to human MCs induced by HG
stimulation in detail in order to explore the underlying molecular
mechanisms involved in the above-mentioned effects of AS-IV. Our
results revealed that following HG stimulation, the levels of
several cellular phosphorylated molecules were decreased by
treatment with AS-IV. Akt, one of the downstream effectors of PI3K,
is involved in cell proliferation and hypertrophy (26). It has been demonstrated that Akt
is activated in renal cells and is regulated by intracellular ROS
(28). In the present study, we
found that the phosphorylation level of Akt was increased by HG
stimulation in MCs. Akt phosphorylation was also markedly reduced
by treatment with AS-IV. Moreover, Akt plays a key role in
promoting the transcriptional activity of NF-κB (29). NF-κB is one of the most important
transcription factors, which can be activated by various stimuli in
DN, such as hyperglycemia, advanced glycation end products (AGEs),
angiogeninII (AngII), oxidative stress and the PI3K/Akt signaling
pathway (49). Akt promotes the
activation of NF-κB by activating the IκB kinase (IKK) to
accelerate the phosphorylation and degradation of IκB, thereby
promoting the translocation of NF-κB from the cytoplasm into the
nucleus and subsequently binding to specific sequences in DNA,
which in turn results in gene transcription. These signaling
cascades eventually lead to MC proliferation (31). Our results revealed that HG
enhanced the activation of NF-κB, and that the degradation and
phosphorylation levels of IκBα were marekdly decreased in the MCs
treated with AS-IV. These data strongly suggest that the Akt/NF-κB
signaling pathway is involved in the pathogenesis of DN.

In addition, we used specific inhibitors of
signaling molecules as a control to compare the effectiveness of
AS-IV in order to validate the upstream and downstream association
among HG-induced NADPH oxidase, Akt and NF-κB activation. The
phosphorylation levels of Akt and IκBα were markedly inhibited by
the NADPH oxidase inhibitor, DPI, or the ROS inhibitor, tempol. Of
note, the PI3K inhibitor, LY294002, also abolished the
HG-stimulated Nox4 expression, as well as IκBα phosphorylation and
degradation. However, the IκBα inhibitor, Sul, did not suppress
Nox4 expression and Akt activation induced by HG. Studies have
indicated that ROS, as an important stimulator of NF-κB activation,
mediate the activation of Akt in MCs and other cultured cells
(50). However, controversially,
there are also data reporting that the intracellular ROS level is
regulated by Akt (26). These
discrepancies suggest the existence of a cross-talk between NADPH
oxidase-derived ROS and Akt activation. Our results support this
assumption and indicate that both the activation of NADPH oxidase
and Akt may be required for HG-induced IκBα phosphorylation and
degradation in MCs.

The early distinctive pathological characteristics
of DN are not only MC proliferation, but also the hypocontractility
of MCs, which is induced by the decreased Ca2+ influx.
TRPC6 is Ca2+ permeable cation channel which plays a
pivotal role in regulating Ca2+ signaling in MCs,
proving a mechanism for impaired MC contraction in diabetes
(10). It has previously been
suggested that the abundance of TRPC6 protein in MCs is decreased
by ROS and PKC in diabetes (51).
Moreover, it has also been reported that NF-κB participates in the
regulation of TRPC6 expression (33). The results of the present study
revealed that exposure to HG resulted in the downregulation of
TRPC6 protein and a reduction in free calcium concentration in the
MCs, inducing the contractile dysfunction of MCs; the NADPH
oxidase/ROS/Akt/NF-κB signaling pathway may also be involved in
these effects, which were markedly supressed by treatment with
AS-IV.

In conclusion, the present study indicates that
hyperglycemia induces glomerular MC proliferation and the
downregulation of TRPC6 protein by promoting Nox4 upregulation, ROS
generation, Akt and NF-κB activation. Treatment with AS-IV inhibits
HG-induced MC proliferation and contractile dysfunction through the
NADPH oxidase/ROS/Akt/NF-κB signaling pathway. Therefore, we
suggest that AS-IV may be a valuable candidate for the prevention
and treatment of early DN. However, other relevant mechanisms
underlying the effects of AS-IV require further investigation.

Acknowledgements

This study was supported by grants from the National
Natural Science Foundation of China (no. 81173624), the Nature
Science Foundation of Anhui Province (no. 11040606M201) and
International scientific and Technological Cooperative Project of
Anhui province (no. 1230603007). The authors would like to thank Li
Gui and Dake Huang from the Synthetic Laboratory of Anhui Medical
University for their helpful technical assistance.